Abstract

Polymorphonuclear leukocytes (PMNs, or neutrophils) are critical for human innate immunity and kill most invading bacteria. However, pathogens such as Staphylococcus aureus avoid destruction by PMNs to survive, thereby causing human infections. The molecular mechanisms used by pathogens to circumvent killing by the immune system remain largely undefined. To that end, we studied S. aureus pathogenesis and bacteria-PMN interactions using strains originally isolated from individuals with community-acquired (CA) and hospital-acquired infections. Compared with strains from hospital infections (COL and MRSA252), strain MW2 and a methicillin-susceptible relative, MnCop, were significantly more virulent in a mouse model of S. aureus infection, and caused the greatest level of pathology in major vital organs. Although phagocytosis of each strain triggered production of reactive oxygen species and granule-phagosome fusion, those from CA infections were significantly more resistant to killing by human PMNs and caused greater host cell lysis. Microarray analysis of the strains during neutrophil phagocytosis identified genes comprising a global S. aureus response to human innate host defense. Genes involved in capsule synthesis, gene regulation, oxidative stress, and virulence, were up-regulated following ingestion of the pathogen. Notably, phagocytosis of strains from CA infections induced changes in gene expression not observed in the other strains, including up-regulation of genes encoding virulence factors and hypothetical proteins. Our studies reveal a gene transcription program in a prominent human pathogen that likely contributes to evasion of innate host defense.

Polymorphonuclear leukocytes (PMNs,3 or neutrophils) are key effectors of the human innate immune system and provide critical defense against invading microorganisms. Phagocytosis of bacteria triggers production of reactive oxygen species (ROS) and release of microbicidal granule components into forming phagosomes. Although PMN-derived antimicrobial products are highly effective at killing most bacteria, some pathogens evade destruction by neutrophils to survive and cause human infections. An enhanced understanding of the molecular mechanisms used by pathogens to circumvent the immune system is essential for developing new treatments for infectious diseases.

Staphylococcus aureus is a leading cause of human infections worldwide. The pathogen causes a variety of diseases including impetigo, cellulitis, food poisoning, toxic shock syndrome, necrotizing pneumonia, endocarditis, and sepsis (1). Risk of S. aureus infection is increased during hospitalization, after surgery or dialysis, and in patients with indwelling percutaneous medical devices and/or catheters (1). Notably, there is an alarming increase in the incidence of community-acquired (CA) S. aureus infections in seemingly healthy individuals, a problem which underscores the need to better understand virulence mechanisms (2). A number of factors are known to contribute to pathogenesis (1, 2, 3, 4, 5, 6); however, a comprehensive analysis of changes in S. aureus gene expression that occur during interaction with the human innate immune system has not been performed. Furthermore, our understanding of the molecular mechanisms used by pathogens to avoid destruction by innate host defense is limited. To that end, we studied S. aureus pathogenesis and global gene expression during phagocytic interaction with human PMNs.

Materials and Methods

Bacterial strains and culture

Staphylococcus aureus strains MW2 (5, 7), MnCop (8), LAC (9, 10, 11, 12), MRSA252 (13), and COL (14) were selected carefully for this study based on several criteria, including the type of human disease caused by each (e.g., MW2 as the prototype for CA methicillin-resistant S. aureus (MRSA), and MRSA252 for its high prevalence in hospitals in the United States and United Kingdom), available genome sequence information (MW2, MRSA252, and COL), historical significance (COL), available genotype information (e.g., MLST, spa type, agr type, and SCCmec type), and methicillin susceptibility (MnCop). Specifically, MW2 (pulsed-field type USA400) was originally isolated from a healthy 16-mo-old girl in North Dakota who died of septicemia and septic arthritis in 1998 (7, 15). LAC (pulsed-field type USA300) has caused recent S. aureus outbreaks in athletes, many of which are skin infections (10, 11, 16). Recent reports also describe USA300 as the causative agent of staphylococcal necrotizing fasciitis (9) and severe community-onset pneumonia in healthy adults (12). MnCop was originally isolated from an 8-mo-old infant who had S. aureus toxic shock syndrome (8). Thus, each of the CA strains used in this study, i.e., MW2, MnCop, and LAC, has been linked to infections in individuals with no known risk factors. In contrast, MRSA252 (pulsed-field type identical to EMRSA16), is the second leading cause of healthcare-associated S. aureus infections in the United States (16). Descriptions of each strain are summarized in Table I⇓.

Mouse model of S. aureus disease

All animal studies conformed to National Institutes of Health guidelines and were approved by the Animal Use Committee at Rocky Mountain Laboratories. Female CD1 Swiss mice were purchased from Charles River. Animals were between 10 and 12 wk of age, housed in microisolator cages, and received food and water ad libitum. S. aureus strains were grown to E1, washed twice with sterile DPBS, and resuspended to 108/100 μl in DPBS. Mice were inoculated with 108S. aureus or with sterile DPBS via the tail vein. The experiment was performed once with 10 mice per S. aureus strain and 5 mice for DPBS control. Criteria for determining morbidity/sickness in mice included hunched posture, decreased activity, ruffled fur, and labored breathing. Animals were euthanized if unable to eat or drink, or if they became immobile. All mice were euthanized by 48 h. Survival statistics were performed with a log-rank test (GraphPad Prism version 4.0 for Windows; GraphPad Software).

To determine whether S. aureus had disseminated to large organs during bacteremia, liver and lung tissues were harvested at time of animal death, homogenized, and aliquots of each homogenate were plated on tryptic soy agar. Colonies were enumerated the following day. Alternatively, parts of the liver, lung, kidney, heart, and brain were collected in parallel for histopathology. Tissues were fixed in 10% neutral-buffered formalin, processed for 12 h in an Excelsior Tissue Processor (ThermoShandon), and dehydrated using a graded series of ethanol. Tissues were then cleared with Histosolve (ThermoShandon) and infiltrated and embedded in paraffin. Paraffin blocks were sectioned at 5 microns and slides were stained with H&E. A trained veterinary pathologist (D. J. Gardner) examined all of the tissue samples. Statistics for dissemination of S. aureus to the liver and lung were performed with a Kruskal-Wallis analysis using Dunn’s posttest for multiple comparisons. Images of organ sections were sharpened and adjusted equally for brightness and contrast with Photoshop CS (Adobe Systems).

Isolation of human PMNs

PMNs were isolated from heparinized venous blood of healthy individuals in accordance with a protocol approved by the Institutional Review Board for Human Subjects, National Institute of Allergy and Infectious Diseases. Briefly, blood was incubated for 20 min at room temperature at a 1:1 ratio with 0.9% sodium chloride (Irrigation USP; Baxter Healthcare) containing 3.0% Dextran T-500 (Amersham Biosciences) to sediment erythrocytes. The leukocyte-containing supernatant was centrifuged at 670 × g for 10 min and resuspended in 35 ml of 0.9% sodium chloride. The cell suspension was underlaid with 10 ml of Ficoll-PaquePLUS (1.077 g/L; Amersham Biosciences) and centrifuged for 25 min to separate PMNs from PBMCs. PBMCs were removed by aspiration and erythrocytes were lysed with water (Irrigation USP; Baxter Healthcare) for 15–30 s followed by immediate mixing with 1.7% sodium chloride. Purified PMNs were centrifuged at 380 × g, resuspended in RPMI/H, and enumerated by microscopy. Purity of PMN preparations and cell viability were assessed by flow cytometry (FACSCalibur; BD Biosciences). Cell preparations contained ∼99% PMNs and all reagents used contained <25.0 pg/ml endotoxin (Limulus Amebocyte Lysate assay; Fisher Scientific).

Phagocytosis of S. aureus by human PMNs

Phagocytosis of S. aureus by human PMNs was determined with fluorescence microscopy as previously described (17). Bacteria were grown to E1, opsonized with 50% normal human serum for 30 min at 37°C, and washed in DPBS. After opsonization, bacteria were labeled with AlexaFluor 488 (Molecular Probes) in DPBS for 20 min at 37°C, and unlabeled fluorochrome was removed by two washes with DPBS. PMNs (3 × 105) were added to serum-coated glass coverslips in 24-well tissue culture plates and allowed to adhere at room temperature for 15 min. Cells were chilled on ice for 10 min and AlexaFluor 488-labeled bacteria were added at a ratio of 10 bacteria:PMN. Plates were then centrifuged at 380 × g for 8 min at 4°C to synchronize phagocytosis (18). Samples were incubated at 37°C in a CO2 incubator for the indicated times (t = 0 was processed immediately after centrifugation), medium was removed from the wells by aspiration, and cells were fixed on ice for 30 min with 4% paraformaldehyde. Fixative was removed by aspiration, and uningested bacteria were counterstained with AlexaFluor594 conjugated-Ab specific for AlexaFluor 488 for 15 min at room temperature. Samples were visualized with a Zeiss Axioskop 2 Plus fluorescence microscope (Carl Zeiss) and imaged with a Zeiss Axiocam digital camera. The total number of S. aureus bound and/or ingested was evaluated in 50 neutrophils per assay from at least five separate fields of view. Percent phagocytosis was calculated with the equation: (number of ingested bacteria per cell/total number of PMN-associated bacteria per cell, bound or ingested) × 100. Statistics were performed with a repeated-measures ANOVA and Tukey’s posttest for multiple comparisons (GraphPad Prism version 4.0 for Windows; GraphPad Software). Images were adjusted for brightness and contrast with Photoshop CS (Adobe Systems).

PMN ROS production

PMN ROS production was measured using a previously described method (18) with minor modifications. PMNs were incubated with 25 μM 2′, 7′-dihydrodiclorofluorescein diacetate (DCF; Molecular Probes) for 20–30 min at room temperature in RPMI/H. DCF-containing PMNs (106) and opsonized S. aureus were combined in wells of a 96-well microtiter plate at 4°C (ratio of 10 bacteria:PMN), centrifuged for 8 min at 380 × g, and transferred to a microplate fluorometer (Spectramax Gemini; Molecular Devices). ROS production was measured continuously at 1-min intervals for up to 180 min at 37°C using excitation and emission wavelengths of 485 and 538 nm, respectively. The rate of PMN ROS production over time (second-order kinetics) were determined from the Vmax (max increase in fluorescence) within each 10-min time period.

PMN bactericidal activity

Killing of S. aureus by human PMNs was determined as described (17) with some modification. Briefly, PMNs (106) were combined with ∼107 opsonized S. aureus in 24-well tissue culture plates, centrifuged at 380 × g for 8 min and incubated at 37°C for up to 6 h. At indicated times, PMNs were lysed with 0.1% saponin (20 min on ice) and S. aureus were plated on tryptic soy agar. Colonies were enumerated the following day, and percent S. aureus survival was calculated with the equation (CFU+PMN at t/CFU+PMN at t0) × 100. The assay measures total number of viable ingested and uningested bacteria. Statistics were performed with a repeated-measures ANOVA and Tukey’s posttest for multiple comparisons (GraphPad Prism).

S. aureus-induced PMN lysis

Following phagocytosis of S. aureus, PMN lysis was determined with a standard assay for release of lactate dehydrogenase (LDH) as described by the manufacturer (Cytotoxicity Detection kit; Roche Applied Sciences). Alternatively, PMN lysis was determined by counting intact cells on a hemacytometer. Statistics were performed with a repeated-measures ANOVA (LDH release) or one-way ANOVA (hemacytometer counts) and Tukey’s posttest for multiple comparisons (GraphPad Prism).

Electron microscopy

For transmission electron microscopy (TEM), phagocytosis assays were performed as described above, but with the following modifications. PMNs (3 × 105) were combined with 3 × 106 serum-opsonized S. aureus in wells of a 24-well tissue culture plate containing serum-coated Thermanox coverslips (Nalge Nunc International). Cells were incubated at 37°C with 5% CO2 for up to 180 min. At the indicated times, cells were fixed in Karnovsky’s fixative containing 4% glutaraldehyde and 4% paraformaldehyde in 0.1 M sodium phosphate buffer, pH 7.2 (Electron Microscopy Sciences). Samples were washed twice in 0.1 M phosphate, and postfixed in 1% osmium/0.8% potassium ferrocyanide in 0.1 M phosphate. Following two additional washes in water, samples were stained in-block with 1% uranyl acetate in water, washed again, and then dehydrated in ethanol, and embedded in Spurr resin (Ted Pella), as described (19). Thin sections were examined on a model H7500 TEM (Hitachi High Technologies) at 80 kV. Images were captured with an Advantage HR digital camera system (Advanced Microscopy Techniques) and adjusted for brightness and contrast with Photoshop CS (Adobe Systems). For scanning electron microscopy, phagocytosis assays were performed as described above, but with the following modifications. Cells were fixed in 0.15 M cacodylate buffer, pH 7.2, containing 2.5% glutaraldehyde and washed twice in cacodylate buffer. Samples were then postfixed with 1.0% osmium tetroxide in cacodylate buffer, washed twice with water, dehydrated with ethanol, and critical-point dried through carbon dioxide. After mounting on stubs, the samples were lightly coated with iridium using an ion beam sputterer (South Bay Technology), and examined with a Hitachi S4500 field emission scanning electron microscope (Hitachi High Technologies America). Digital images were collected with an Orion system (Focused Resolutions), and adjusted for brightness and contrast with Photoshop CS (Adobe Systems).

Analysis of S. aureus gene expression

To compare transcript levels in vitro, S. aureus strains were cultured to the desired phase of growth and ∼2.5 × 109 cells were lysed with 700 μl of RLT buffer (Qiagen). Lysates were homogenized with an FP120 FastPrep system (QBiogene). Total RNA was isolated with RNeasy kits (Qiagen) and contaminating chromosomal DNA was removed by DNase treatment (Qiagen). Purified bacterial RNA was used to prepare fragmented and biotin-dUTP-labeled cDNA according to standard Affymetrix prokaryotic target preparation (〈www.affymetrix.com/support/downloads/manuals/expression_s3_manual.pdf〉). Briefly, cDNA was synthesized from ∼10 μg of total RNA in 1× first-strand buffer containing 25 ng/μl random primers (Invitrogen Life Technologies), 10 mM DTT, 0.5 mM dNTPs (Amersham), 0.5 U/μl SUPERase In (Ambion), and 25 U/μl SuperScript II (Invitrogen Life Technologies). Remaining RNA was removed by hydrolysis with 1 N NaOH and the reaction was neutralized with 1 N HCL. cDNA (∼3–6 μg/sample) was fragmented using ∼0.6 U of DNase I (Amersham Pharmacia Biotech) per μg of cDNA in One-Phor-All Buffer (Amersham). The 3′ termini of the fragmented cDNA was labeled using an Enzo BioArray Terminal Labeling kit with Biotin-dUTP (Enzo Diagnostics). The reaction was completed as suggested by Affymetrix using the Midi format. Biotinylated S. aureus cDNA was hybridized to custom Affymetrix GeneChips (RMLChips) containing 3961 probe sets from eight different S. aureus strains (COL, EMRSA16, MSSA476, RF122, TSS.8325, Mu50, and N315) and scanned according to standard GeneChip protocols (Affymetrix). Specific details for Affymetrix hybridization and scanning protocols can be found at the internet address provided above. Each experiment was repeated in triplicate. Data were analyzed with GeneChip Operating Software version 1.1 (Affymetrix) and GeneSpring version 6.0 (Silicon Genetics). Data was normalized using the mean signal from the S. aureus probe sets multiplied by a scale factor to obtain the target signal. Default detection thresholds of p = 0.04 and p = 0.06 were used to determine genes that were present and absent calls were assigned to values of p ≥ 0.06. The Pearson correlation (GeneSpring) was used to evaluate the replicate correlation. Any nongrouping replicates were deleted from further evaluation. Quality filters based upon Present-Absent calls and signal intensities were used to create final gene lists (Microsoft Excel 2003). Microarray data are posted on the Gene Expression Omnibus (GEO, 〈www.ncbi.nlm.nih.gov/geo/〉, accession number GSE2728).

To measure S. aureus gene expression during S. aureus–PMN interaction, bacteria were grown to E1, washed once in DPBS, and opsonized in 50% normal human serum at 37°C for 30 min. PMNs (107) were combined with ∼108 opsonized S. aureus in wells of a 12-well tissue culture plate on ice and centrifuged at 380 × g for 10 min at 4°C to synchronize phagocytosis (17, 18). Plates were transferred to a 37°C incubator with 5% CO2 for the desired times and samples were lysed as described above. For the microarray data derived from S. aureus-PMN interaction, RNA was isolated from the combination of three populations of bacteria: 1) S. aureus bound to PMNs; 2) S. aureus ingested by PMNs; and 3) free (uningested) S. aureus. However, we determined in separate experiments using fluorescence microscopy, and transmission and scanning electron microscopy, that there were few, if any, free bacteria after 30 min of S. aureus-PMN interaction (see Fig. 2⇓, and data not shown). Further, after 60 min of S. aureus-PMN interaction, typically all neutrophil-associated bacteria were internalized (data not shown). At desired times, PMNs and bacteria in each well were lysed with 700 μl of RLT buffer (Qiagen). Isolation of S. aureus RNA and microarray analyses were performed as described above, except that fold-changes for each gene were determined by comparing RMLChips hybridized with cDNA from S. aureus alone to those with cDNA from S. aureus during PMN phagocytosis (time-matched). To be included in the final gene lists (e.g., see Figs. 5–9⇓⇓⇓⇓⇓, and supplemental Table V4), genes must have met the quality filters described above, and changes in gene expression must have been at least 2-fold in one of the strains. For those genes (>2-fold change in one strain), changes of >1.5-fold were also indicated for the other strains. PMN-S. aureus microarray experiments were performed three separate times with PMNs from three blood donors. Although the RMLChip was designed to avoid cross-hybridization with human RNA or DNA, we determined empirically (using neutrophils from three individuals) that PMN RNA failed to hybridize with RMLChips. In our experiments, 3957 of 3961 probe sets had no significant cross-hybridization with PMN RNA. The four probe sets with noted binding to neutrophil RNA were eliminated from the analysis.

TaqMan real-time RT-PCR analysis

Phagocytosis experiments and RNA preparation for TaqMan analysis were performed with procedures and conditions identical to those used for the PMN-S. aureus microarray experiments. TaqMan real-time RT-PCR analysis of two separate phagocytosis experiments (each assayed in triplicate) using PMNs from two human blood donors was performed with an ABI 7500 thermocycler (Applied Biosystems). Relative quantification of S. aureus genes was determined by the change in expression of target transcripts relative to gyrB (housekeeping or calibration gene) according to the manufacturer’s protocol (Applied Biosystems Relative Quantification Manual at 〈http://docs.appliedbiosystems.com/genindex.taf〉, doc. index no. 4347824).

Results

S. aureus virulence in a mouse infection model

To gain new insight into S. aureus pathogenesis, we tested virulence of strains originally isolated from individuals with CA (MW2, MnCop, and LAC) and hospital-acquired (HA; COL and MRSA252) infections in a mouse model of bacteremia (Table I⇑, and Fig. 1⇓). All mice infected with MW2, MnCop, or LAC were moribund or noticeably sick (10 of 10 animals infected with each strain, see Materials and Methods for details). In contrast, those infected with COL or MRSA252, a strain reported as highly virulent in humans (13), appeared relatively healthy (0 of 10 animals infected with COL and 1 of 10 of those infected with MRSA252 were sick). Correspondingly, mortality was significantly increased in those infected with MW2 or MnCop (p = 0.015, log-rank test) (Fig. 1⇓A). At the time of death, major vital organs were evaluated for signs of infection (Fig. 1⇓, B–E). On average, more bacteria were recovered from the livers of animals infected with MW2, MnCop, or LAC compared with the other strains (Fig. 1⇓B). There were also a greater number of S. aureus recovered from lungs of mice infected with MW2 or MnCop compared with the other strains (Fig. 1⇓C). These observations are consistent with the known capacity of MW2 and similar strains to cause necrotizing pneumonia in healthy individuals (20).

S. aureus pathogenesis. A, Mouse survival. Mice were infected with S. aureus strains by i.v. inoculation (tail vein), and health was monitored for 48 h. Results are from 10 mice in each infection group and five mice in a PBS control group. ∗, p = 0.015, curves are different. B and C, Dissemination of S. aureus. Homogenates from the liver and lung of animals infected as described in A were plated for CFUs. ∗, p < 0.05 vs PBS control mice. D–F, Vital organ pathology in infected mice. Heart (D), kidney (E), and brain tissue (F) from several animals was examined for disease. Yellow arrows indicate acute neutrophilic inflammation, bacterial colonies (solid purple matter), and/or regions of necrosis. Black squares, regions magnified ×20.

We next performed histopathology on selected organs to determine the extent of disseminated disease caused by each strain (Fig. 1⇑, D–F). Heart tissues from animals infected with MW2, MnCop, or LAC had multifocal areas of necrosis and acute (neutrophilic) inflammation with bacterial colonies (Fig. 1⇑D). Infection also extended to kidneys and brain in mice infected with MW2 or MnCop, providing further evidence for enhanced virulence in these strains (3 of 7 animals tested had diseased brain or spinal cord tissue) (Fig. 1⇑, E and F). In contrast, organs from animals infected with COL or MRSA252 typically showed little or no overt signs of disease (Fig. 1⇑, D and E, and data not shown). These results indicate that strains MW2, MnCop, or LAC are generally more virulent than either MRSA252 or COL. Inasmuch as PMNs are essential effectors of innate host defense against bacterial pathogens, the enhanced virulence of MW2, MnCop, and LAC in the mouse model may reflect increased resistance to neutrophil killing. This hypothesis is tested below.

Phagocytosis and killing of S. aureus by human PMNs

To determine whether differences in virulence are due in part to varied capacity of the strains to interact with the innate immune system, we evaluated phagocytosis and killing of each by human neutrophils. Although there were differences in early uptake of the strains (e.g., ingestion of COL and MW2 at 5 min was 65.1 ± 7.9% and 46.3 ± 12.9%, respectively, p < 0.05, ANOVA with Tukey’s posttest), phagocytosis of each strain occurred rapidly and was essentially complete by 15 min (Fig. 2⇓, A and B). Notably, uptake of each strain by PMNs was virtually identical by 60 min of S. aureus-PMN interaction and there were few or no free bacteria (Fig. 2⇓, and data not shown). We next determined that ingested S. aureus were exposed to microbicidal PMN ROS and granule contents (Fig. 2⇓, C–E). Despite the exposure to neutrophil antibacterial components, there was significant S. aureus survival after phagocytosis (Fig. 2⇓F), findings consistent with studies by Gresham et al. (21). Further, survival was significantly better for strains isolated from patients with CA infections (e.g., survival at 30 min was 58.7 ± 7.6% for MW2 vs 32.0 ± 3.0% for MRSA252, p < 0.001, ANOVA with Tukey’s posttest) (Fig. 2⇓F). These observations provide strong support to the notion that S. aureus infections are caused at least in part by evasion of neutrophil-mediated killing.

Gene expression in S. aureus strains from CA and HA infections

As a first step toward understanding the molecular basis of differences in virulence and interaction with neutrophils, we compared global gene expression in the strains grown in vitro with S. aureus microarrays (Table I⇑, Fig. 4⇓, and supplemental Tables II-IV). Collectively, expression of 210 genes differentiated MW2, MnCop, and LAC from MRSA252 and COL (supplemental Table II). Only 17 genes (not expressed in strains COL or MRSA252) were commonly expressed in the three CA strains during growth in vitro (supplemental Table II). Nine of the 17 genes encoded hypothetical proteins, and 3 others were phage-encoded (supplemental Table II). The finding that MW2, MnCop, and LAC collectively express a limited number of unique transcripts (not present in COL or MRSA252) is consistent with the genetic similarity between LAC and COL based on MLST and spa typing, and gene expression (Fig. 4⇓C, and Table I⇑). Although strain-specific gene expression (Fig. 4⇓) might underlie differences in pathogenesis, it is likely that many pathogen processes used to evade PMN killing are triggered by interaction with the innate immune system.

Global gene expression in S. aureus strains of diverse genetic backgrounds. A–C, Comparative microarray analysis of S. aureus strains cultured in vitro was measured with custom Affymetrix microarrays containing 3961 open reading frames from eight S. aureus genomes. Genes were included in the analysis if they were called “present” at the phase of growth indicated. A, Gene expression in MW2, COL, and MRSA252. B, Gene expression in MW2, MnCop, and LAC. C, Gene expression in LAC, COL, and MRSA252. Results are the average of three separate experiments for each strain. Phases of growth are as follows: EE, early-exponential (OD600 = 0.4); E1, mid-exponential (OD600 = 0.75); E2, mid-exponential (OD600 = 1.0); ES, early-stationary. The individual genes from which these comparisons were made are provided in supplemental Tables II–IV.

PMN phagocytosis induces global changes in S. aureus gene expression

To further elucidate the molecular mechanisms used by the pathogen to evade innate host defense, we measured global changes in S. aureus gene expression during phagocytic interaction with human PMNs. We discovered that 21.8–39.1% of the genes in S. aureus were differentially regulated at any given time following phagocytic interaction (e.g., 593, 857, and 1029 genes in COL, MRSA252, and MW2, respectively, were up- or down-regulated at 180 min after phagocytosis) (Figs. 5–9⇓⇓⇓⇓⇓, and supplemental Table V). Genes differentially expressed during PMN phagocytosis were divided into categories based on function or annotation. Genes encoding proteins with undefined function and those involved in metabolism comprised the two largest categories of S. aureus genes modulated by PMN phagocytosis (e.g., n = 348 and 372 genes, respectively, for MW2 30 min after initial PMN interaction) (Figs. 5⇓ and 8⇓, and supplemental Table V). Notably, 26.8–38.8% of S. aureus genes were differentially regulated within 30 min after PMN phagocytosis, depending on the strain analyzed (e.g., 26.8% of those in COL and 38.8% of those in MW2) (Fig. 5⇓).

Global changes in S. aureus gene expression during phagocytosis. S. aureus gene expression was measured following PMN phagocytosis as described in Materials and Methods. The number of S. aureus genes up (red bars)- or down (blue bars)-regulated at 30, 60, or 180 min after phagocytosis is indicated for assigned functional categories for each strain. The percent genes changed is based on the total number of possible coding regions in each sequenced strain (i.e., MW2, 2632 genes; MRSA252, 2673 genes; COL, 2721 genes).

S. aureus genes differentially expressed during PMN phagocytosis. Results are presented as the mean fold-increase or -decrease of three separate experiments with PMNs from three different blood donors (comparison is with time-matched assays containing only opsonized S. aureus). White asterisks indicate genes called “absent” before (for induced genes) or after (for repressed genes) interaction with neutrophils. ∗∗, Gene expression data from two probe sets was used to generate the information as displayed. An extended set of these data are published as supplemental Table V.

Phagocytosis triggers differential regulation of S. aureus genes involved in cell division, replication, and biosynthetic activity. Gene expression was determined as described in the legend of Fig. 6.

Genes encoding proteins that moderate oxidative stress, and those involved in metabolism and capsule synthesis are induced during phagocytosis

Consistent with the finding that S. aureus was exposed to PMN-derived ROS (Fig. 2⇑C), at least 25 stress response genes were up-regulated in the pathogen during phagocytosis (Fig. 6⇑, and supplemental Table V). For example, genes encoding catalase (katA), thioredoxin (trxA), thioredoxin reductase (trxB), superoxide dismutase (sodA, sodM), alkyl hydroperoxide reductase (ahpC, ahpF), and glutathione peroxidase (gpxA), were up-regulated in S. aureus immediately following phagocytosis (at 30 and 60 min), a period of time that corresponds with maximum generation of neutrophil ROS (Figs. 2⇑C and 6⇑). Genes encoding proteins involved in capsular polysaccharide synthesis (capA-G, capI-P, and cap8I) were up-regulated in all but one of the strains during initial S. aureus-PMN interaction (Fig. 6⇑, and supplemental Table V). Although there is evidence that capsule blocks phagocytosis (23), under our conditions each strain was readily ingested by neutrophils (Fig. 2⇑A). Thus, the data suggest S. aureus capsule is important for survival once the pathogen is internalized. Alternatively, induction of S. aureus capsule by PMN uptake might confer a phagocytosis-resistant phenotype to bacteria that escape destruction from initial neutrophil interaction.

Discussion

S. aureus is one of the most prominent human pathogens and is responsible for diverse infections worldwide (1). Risk of S. aureus infection is increased during or after hospitalization, and there is a serious problem with antibiotic resistance in the pathogen in healthcare settings (1, 2). In contrast, there has been a dramatic increase in the number of S. aureus infections occurring in healthy individuals with no apparent risk factors for disease (2, 7, 9, 10, 12, 15, 36, 37, 38, 39, 40, 41, 42, 43). The molecular basis for these infections is largely unknown, although it is likely that a combination of pathogen virulence mechanisms and host susceptibility factors contribute to disease.

Neutrophils are a critical component of innate immunity and are essential for controlling bacterial infections (44, 45, 46, 47, 48, 49, 50). Defects that alter normal PMN function, such as chronic granulomatous disease and leukocyte adhesion deficiency, predispose individuals to serious S. aureus infections (reviewed in Ref.44). Further, it has long been known that individuals with neutropenia are more susceptible to bacterial infections, including those caused by S. aureus (45, 46, 47, 48). Recent studies in animal models of S. aureus disease support these observations unambiguously (49, 50). Inasmuch as evasion of innate immunity plays a critical role in bacterial pathogenesis (17, 19, 51), a significant part of our study investigated the interaction of human PMNs with S. aureus. Of the S. aureus strains used in our study, those originally isolated from individuals with CA infections (no established risk factors) evaded killing by human neutrophils better than those isolated from hospital infections (Table I⇑ and Fig. 2⇑F). Differences in pathogen survival were not due to altered neutrophil function, because phagocytosis, ROS production and degranulation were ultimately comparable for each of the strains tested (Fig. 2⇑). Rather, the data indicate that strains MW2, MnCop, and LAC are more resistant to destruction per se and/or produce factors that cause more rapid host cell lysis by comparison (Fig. 3⇑). Notably, there was no net growth by any of the strains during phagocytic interaction with human neutrophils until sometime after 3 h of culture (Fig. 2⇑F). The increased survival/growth by MW2, MnCop, and LAC at 6 h was reflected generally by the degree of PMN lysis caused by the strains (compare Fig. 2⇑F with Fig. 3⇑, A and B). Thus, our data indicate that some S. aureus survived intraphagosomally without multiplying, but produced factors that cause PMN lysis.

Results from the neutrophil-S. aureus experiments were paralleled by the mouse pathogenesis data, which indicate MW2, MnCop, and LAC (those from community infections) are more virulent than COL or MRSA252 (Fig. 1⇑), consistent with recent studies indicating strain-to-strain variability in S. aureus virulence (52). Strains LAC (pulsed-field type USA300) and MW2 (pulsed-field type USA400) are MRSA strains closely associated with community outbreaks of S. aureus disease in the United States (5, 7, 9, 10, 12, 36, 38, 41, 53). For example, recent reports indicate USA300 is a causative agent of staphylococcal necrotizing fasciitis (9) and severe community-onset pneumonia in healthy adults (12). In contrast, MRSA252 is a leading cause of hospital infections in the United States and United Kingdom (16), but to our knowledge is not a typical source of disease in individuals without associated risk factors. We note also that methicillin resistance has little to do with virulence per se, because MnCop, an MSSA strain closely related to MW2, was similar to MW2 in the mouse model of infection and in each of our host-pathogen assay systems (Figs. 1–3⇑⇑⇑, 6⇑, and 7⇑). Taken together, our findings provide strong support to the idea that enhanced strain virulence is linked to (or results from) evasion of killing by neutrophils, which likely underlies the ability of MW2, MnCop, and LAC to cause disease in individuals without known risk factors.

Several observations support the idea that the global pathogen response described herein is used by S. aureus to circumvent innate immunity and thereby promote disease in humans. Notably, global changes in S. aureus gene expression were triggered by PMN phagocytosis, and neutrophils are the predominant host cells used in defense against bacterial infections. Immediate (within 30 min) up-regulation of genes involved in virulence, metabolism, capsule synthesis, and gene regulation after ingestion by neutrophils correlated with S. aureus survival, which was ≥30% for all strains within the first 90 min after phagocytosis (≥44% for MW2, MnCop, and LAC) (Figs. 2⇑F and 6⇑). Given the noted increases in expression of ahpC, ahpF, katA, gpxA, sodA, sodM, trxA, and trxB after phagocytosis, the strains used in this study are probably not susceptible to destruction by primary neutrophil-derived reactive oxygen species, i.e., superoxide and hydrogen peroxide. Preliminary studies by our laboratory have shown that there is ≥60% survival for each of the S. aureus strains after culture with up to 50 mM hydrogen peroxide (K. Braughton and F. DeLeo, unpublished observations).

One of the highlights of our study is that several S. aureus gene regulatory systems were induced after interaction with PMNs. We previously identified a pathogen survival response in group A Streptococcus (GAS, Streptococcus pyogenes) triggered by neutrophils through ihk-irr, a two component gene regulatory system required for virulence (19, 51). The ihk-irr survival response is activated by exposure to neutrophil α-granule components and reactive oxygen species (51). Because there is significant S. aureus survival after interaction with neutrophils, a similar as yet undefined mechanism must exist in this pathogen. As such, many of the genes reported herein likely comprise a S. aureus pathogen survival response controlled directly or indirectly by one or more of the gene regulatory systems induced by phagocytosis. Consistent with this notion, Gresham et al. (21) reported that survival of S. aureus within PMNs was linked to a global gene regulator called sar, and sarA was up-regulated in all S. aureus strains following PMN phagocytosis in our present work (Fig. 6⇑). Taken together, these findings are consistent with the idea that sarA is important for evasion of killing by neutrophils. A systematic investigation of each of the gene regulators identified in our study by targeted gene mutagenesis and/or knockout studies will be necessary to elucidate the role of each in pathogenesis.

Significant emphasis has been placed on determining principal virulence factors responsible for the spread of CA S. aureus infections. The Panton-Valentine leukocidin (encoded by lukF-PV and lukS-PV, or simply pvl) has been linked to CA infections resulting from methicillin-resistant S. aureus (20, 36, 38, 39, 54). Although our studies failed to address pvl expression in S. aureus during PMN phagocytosis, a recent study by Said-Salim et al. (55) demonstrated that there is no correlation between neutrophil lysis and presence of pvl. Importantly, our work identified many other genes potentially responsible for the differences in PMN lysis and strain virulence, and thus, a possible explanation for the increased virulence of CA strains such as MW2 (Figs. 1⇑, 6⇑, and 7⇑, and supplemental Table V). For example, several putative membrane or exported proteins of uncharacterized or unknown function were up-regulated only in strains that cause CA infections (Figs. 6⇑ and 7⇑, and supplemental Table V). These genes clearly merit further investigation.

An enhanced understanding of the molecular mechanisms used by S. aureus to circumvent the human innate immune system will facilitate development of new treatments for bacterial infections. Our previous work with the prominent human pathogen GAS indicated that ∼16% of its genes are differentially regulated during phagocytic interaction with neutrophils (19). By comparison, we determined that up to ∼39% of the genes in S. aureus changed following phagocytosis (Fig. 5⇑, see MW2 at 180 min). Differences in the sensitivity of the methods used (spotted vs oligonucleotide microarrays) may account for some of the differences. However, the two pathogens are quite distinct with regard to genome size, their interaction with human neutrophils, and in the types of disease caused by each. For example, GAS produces numerous molecules that inhibit PMN phagocytosis (reviewed in Ref.56), whereas S. aureus is readily ingested. Nonetheless, each pathogen uses strategies to prolong intraphagosomal survival and cause eventual host cell lysis (Figs. 2⇑ and 3⇑) (19, 51, 56). Thus, there likely exist some common molecular mechanisms for immune evasion between the two pathogens.

We used microarrays to generate a global view of the genes and gene regulatory networks involved in bacterial pathogenesis and therefore generated a vast repository of information from which many new studies of S. aureus pathogenesis will emerge. The global pathogen response revealed by our studies identified dozens of potential vaccine Ags and targets for therapeutics designed to control S. aureus infections.

Acknowledgments

We are grateful to Michael Otto and Scott Kobayashi for critical review of the manuscript and Layre Parkins for technical assistance.

Disclosures

The authors have no financial conflict of interest.

Footnotes

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

↵1 This work was supported in part by the Intramural Research Program of the National Institutes of Health, National Institute of Allergy and Infectious Diseases.

From the Centers for Disease Control and Prevention. 1999. Four pediatric deaths from community-acquired methicillin-resistant Staphylococcus aureus–Minnesota and North Dakota, 1997–1999. J. Am. Med. Assoc.282:1123.-1125.